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. 2016 Jun 10;90(13):5886-5898.
doi: 10.1128/JVI.00262-16. Print 2016 Jul 1.

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes

Rémi Planès  1   2 , Nawal Ben Haij  1   2 , Kaoutar Leghmari  1   2 , Manutea Serrero  1   2 , Lbachir BenMohamed  3   4 , Elmostafa Bahraoui  5   2
Affiliations

HIV-1 Tat Protein Activates both the MyD88 and TRIF Pathways To Induce Tumor Necrosis Factor Alpha and Interleukin-10 in Human Monocytes

Rémi Planès et al. J Virol. .

Abstract

In this study, we show that the HIV-1 Tat protein interacts with rapid kinetics to engage the Toll-like receptor 4 (TLR4) pathway, leading to the production of proinflammatory and anti-inflammatory cytokines. The pretreatment of human monocytes with Tat protein for 10 to 30 min suffices to irreversibly engage the activation of the TLR4 pathway, leading to the production of tumor necrosis factor alpha (TNF-α) and interleukin-10 (IL-10), two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Therefore, this study analyzed whether the HIV-1 Tat protein is able to activate these two pathways separately or simultaneously. Using three complementary approaches, including mice deficient in the MyD88, TIRAP/MAL, or TRIF adaptor, biochemical analysis, and the use of specific small interfering RNAs (siRNAs), we demonstrated (i) that Tat was able to activate both the MyD88 and TRIF pathways, (ii) the capacity of Tat to induce TIRAP/MAL degradation, (iii) the crucial role of the MyD88 pathway in the production of Tat-induced TNF-α and IL-10, (iv) a reduction but not abrogation of IL-10 and TNF-α by Tat-stimulated macrophages from mice deficient in TIRAP/MAL, and (v) the crucial role of the TRIF pathway in Tat-induced IL-10 production. Further, we showed that downstream of the MyD88 and TRIF pathways, the Tat protein activated the protein kinase C (PKC) βII isoform, the mitogen-activated protein (MAP) kinases p38 and extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-κB in a TLR4-dependent manner. Collectively, our data show that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10.

Importance: In this study, we demonstrate that by recruiting the TLR4 pathway with rapid kinetics, the HIV-1 Tat protein leads to the engagement of both the MyD88 and TRIF pathways and to the activation of PKC-βII, MAP kinase, and NF-κB signaling to induce the production of TNF-α and IL-10, two cytokines strongly implicated in the chronic activation and dysregulation of the immune system during HIV-1 infection. Thus, it may be interesting to target Tat as a pathogenic factor early after HIV-1 infection. This could be achieved either by vaccination approaches including Tat as an immunogen in potential candidate vaccines or by developing molecules capable of neutralizing the effect of the Tat protein.

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Figures

FIG 1
FIG 1
The Tat protein induces TNF-α and IL-10 production in human monocytes in a specific manner. (A) Human monocytes were incubated with increasing amounts of full-length HIV-1 Tat protein, either recombinant (Tat), chemically synthesized (synthetic Tat), or acetylated at lysine 50 (K50). As a control, the recombinant Tat protein (10 nM) or LPS (100 ng/ml) was pretreated with trypsin for 1 h at 37°C or kept at 37°C for 1 h without trypsin. After 24 h of treatment, levels of TNF-α (gray bars) and IL-10 (black bars) in the supernatants of the cells were quantified by ELISA. Unst., unstimulated cells. (B) Human monocytes were incubated with HeLa-Tat cells (Delta20 line) stably transfected with tat-rev-vpu gene-conditioned medium (100 μl) in the absence or presence of a mixture of three monoclonal antibodies (1 μg or 10 μg/ml), recognizing epitopes localized in domains 1-15, 46-60, and 78-86 of the Tat sequence, or 10 μg/ml of a monoclonal antibody against HIV-1 gp120 (Mab110-4), which recognizes an epitope within the V3 region. As positive controls, human monocytes were treated with either recombinant Tat (10 nM) or LPS (10 ng/ml). After 24 h, the cell supernatants were collected, and TNF-α (gray bars) and IL-10 (black bars) levels were quantified by ELISA. The data represent means and SD for three independent experiments. Statistical significance was analyzed by one-way ANOVA follow by Bonferroni posttests and is marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant. All bars were compared to those for untreated cells unless otherwise specified (indicated with a black line above the bars).
FIG 2
FIG 2
Tat activates the TLR4 pathway with rapid kinetics. Human monocytes were incubated with 10 nM full-length HIV-1 Tat protein (Tat 1-86) in the presence or absence of various concentrations of anti-TLR4 antibodies as described below. After 24 h of treatment, levels of TNF-α (gray bars) and IL-10 (black bars) in the supernatants of the cells were quantified by ELISA. (A) (Dose-response) Cells were first preincubated with escalating amounts of anti-TLR4 (0.01 to 1 μg/ml) for 60 min before treatment with Tat. (Competition) Anti-TLR4 antibodies (0.01 to 1 μg/ml) and Tat protein (10 nM) were added to the cells at the same time. (Kinetic) Cells were first stimulated with Tat (10 nM) for 5 to 60 min before anti-TLR4 MAbs were added at 0.1 μg/ml or 1 μg/ml. (B) Cells (106 human monocytes) were first preincubated with 0.1 or 1 μg/ml of anti-TLR4 blocking antibodies for 60 min, washed twice or left unwashed, and then stimulated with Tat (10 nM). Cytokine production levels in cell supernatants were quantified after 24 h of treatment. The data represent means and SD for three independent experiments.
FIG 3
FIG 3
HIV-1 Tat protein induces TNF-α and IL-10 production via TLR4-, MyD88-, and TRIF-dependent pathway. Primary mouse peritoneal macrophages isolated from WT (black bars), TLR4−/− (dark gray bars), MyD88−/− (gray bars), TRIF−/− (light gray bars), and TIRAP/MAL−/− (white bars) mice were incubated with increasing amounts of recombinant GST-Tat (full length; Tat 1-101) or the GST-tagged N-terminal fragment (Tat 1-45; carrying the first 45 amino acids) or with an equal amount of GST alone. LPS (100 ng/ml; TLR4 ligand) and Pam3CSK4 (100 ng/ml; TLR1/2 ligand) were used as positive controls. After 20 h of incubation, mouse TNF-α (A) and mouse IL-10 (B) levels in the cell supernatants were quantified by ELISA. The data represent means and SD for three independent experiments. The statistical significance of differences between different groups (indicated with black lines above the bars) was analyzed by two-way ANOVA followed by Bonferroni posttests and is marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.
FIG 4
FIG 4
HIV-1 Tat protein leads to time-dependent and proteasome-dependent TIRAP/MAL degradation via its N-terminal domain. THP-1 cells (106 cells/condition) were treated with recombinant GST-Tat (full-length; Tat 1-101) (A) or the GST-tagged N-terminal fragment (Tat 1-45) (B) at 180 nM. (C) Cells were preincubated with MG132 (20 μM) for 60 min before treatment with Tat 1-45. As positive controls, cells were treated with LPS (1 μg/ml) (D) and Pam3CSK4 (1 μg/ml) (E). Cells were lysed after different times (0 to 90 min). Cellular proteins were separated by SDS-PAGE and analyzed by Western blotting for TIRAP/MAL and IκBα expression. Actin was also quantified as a loading control. Quantification of the bands obtained from 3 independent experiments was performed using ImageJ software. Data represent TIRAP/MAL expression or IκB expression normalized to actin expression.
FIG 5
FIG 5
HIV-1 Tat protein activates NF-κB downstream of TLR4-, MyD88-, TIRAP/MAL-, and TRIF-dependent pathways. (A) Primary mouse peritoneal macrophages isolated from WT, TIRAP/MAL−/−, or TRIF−/− mice were incubated with LPS (10 ng/ml) or the full-length Tat protein (100 nM) or left untreated (−) for 30 min. Cells were lysed, and the p65 subunit of NF-κB was detected in the nuclear fraction of the cells by Western blotting. TFIIB and tubulin were used as loading controls. Quantification of the bands obtained from 3 independent experiments was performed using ImageJ software. Data represent NF-κB (p65) expression in the nucleus normalized to TFIIB expression. (B) HEK cells expressing TLR4/MD2-CD14 were cotransfected with an NF-κB reporter plasmid (SEAP) together with the same amount of pORF-LacZ. Cells were also cotransfected with the indicated amounts (in micrograms) of plasmids encoding siRNAs targeting TLR4, TLR2, MyD88, and TIRAP/MAL or control siRNA or were left without transfection with siRNA (white bar, mock transfection; black bar, Tat stimulation). After 24 h of transfection, cells were left untreated (mock) or treated with Tat 1-45 (100 nM). Twenty-four hours after stimulation, NF-κB-driven SEAP reporter gene expression in the culture supernatants was measured. For normalization, cells were lysed and expression of β-galactosidase was quantified. The data represent means and SD for three independent experiments. Statistical significance was analyzed by one-way ANOVA followed by Bonferroni posttests and is marked as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant. All bars were compared to Tat-treated cells in the absence of cotransfection with any siRNA (black bars) unless otherwise specified.
FIG 6
FIG 6
HIV-1 Tat protein activates NF-κB, MAP kinases ERK1/2 and p38, and PKC-βII in a TLR4-dependent manner. (A and B) Primary human monocytes were incubated with Tat protein (100 nM) in the presence or absence of anti-TLR4 antibodies (1 μg/ml). LPS (100 ng/ml) treatment was used as a positive control for TLR4 pathway activation, and PMA (100 ng/ml) was used as a positive control for PKC activation. After 30 min, cells were lysed as described in Materials and Methods to prepare cytoplasm/membrane (A) or cytoplasm/nucleus (B) protein extracts. (A) Membrane (M) and cytoplasmic (C) protein extracts were separated by SDS-PAGE and analyzed by Western blotting using specific antibodies for PKC isoforms (PKC-βII and PKC-δ). Quantification of the membrane/cytoplasm signal ratios for 3 independent experiments was performed using ImageJ software. The histogram shows the ratios of membrane/cytoplasm PKC-βII (black bars) and PKC-δ (gray bars). Detection of tubulin and Na+/K+ ATPase were performed to control for the quality of protein extracts from the cytoplasmic and membrane compartments, respectively. (B) Cytoplasmic and nuclear extracts were separated by SDS-PAGE and analyzed by Western blotting using specific antibodies for NF-κB/p65, ERK1/2, phospho-ERK1/2, p38, and phospho-p38. Cytoplasmic and nuclear fractions were normalized by use of anti-actin and anti-TFIIB, respectively. Quantification of the bands obtained from 3 independent experiments was performed using ImageJ software.
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